DISSERTATION submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Identification and functional characterization of candidate genes in recurrently gained genomic regions of mantle cell lymphoma and chronic lymphocytic leukemia presented by Diplom-Biologin Alexandra Farfsing born in Iserlohn Heidelberg 2009 Accepted by the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany: May 12, 2009 Referees: Prof. Dr. Werner Buselmaier Prof. Dr. Peter Lichter Day of the oral examination: July 02, 2009 The investigations of the following dissertation were performed from February 2006 till January 2009 under the supervision of Prof. Dr. Peter Lichter and Dr. Armin Pscherer in the division of molecular genetics at the German Cancer Research Center (DKFZ), Heidelberg, Germany. Publication Farfsing A, Engel F, Seiffert M, Hartmann E, Ott G, Rosenwald A, Stilgenbauer S, Döhner H, Boutros M, Lichter P, Pscherer A Gene knockdown studies identified CCDC50 as candidate gene in mantle cell lymphoma and chronic lymphocytic leukemia In preparation. Declarations I hereby declare that I have written the submitted dissertation ‘Identification and functional characterization of candidate genes in recurrently gained genomic regions of mantle cell lymphoma and chronic lymphocytic leukemia’ myself and in this process have used no other sources or materials than those expressly indicated. I hereby declare that I have not applied to be examined at any other institution, nor have I used the dissertation in this or any other form at any other institution as an examination paper, nor submitted it to any other faculty as a dissertation. _____________________________ _____________________________ (Place, Date) Alexandra Farfsing To Jan & my family Contents Contents Abbreviations ................................................................................................................................................. iii Summary.......................................................................................................................................................... v Zusammenfassung .......................................................................................................................................... vi 1 Introduction ............................................................................................................................................ 1 1.1 Cancer ............................................................................................................................................ 1 1.1.1 The hallmarks of cancer ................................................................................................... 1 1.1.2 Oncogenes ....................................................................................................................... 2 1.1.3 Tumor suppressor genes .................................................................................................. 3 1.2 Leukemia ....................................................................................................................................... 4 1.2.1 Acute Leukemia ................................................................................................................ 4 1.2.2 Chronic Leukemia ............................................................................................................. 4 1.3 B-cell neoplasms ............................................................................................................................ 5 1.3.1 B-cell development .......................................................................................................... 6 1.3.2 The cellular origin of human B-cell lymphomas ............................................................... 8 1.4 Mantle cell lymphoma ................................................................................................................... 9 1.4.1 Oncogenic mechanisms in MCL ....................................................................................... 9 1.4.2 Secondary genetic alterations ........................................................................................ 10 1.5 B-cell chronic lymphocytic leukemia ........................................................................................... 11 1.5.1 Oncogenic mechanisms in CLL ....................................................................................... 11 1.5.2 Secondary genetic alterations ........................................................................................ 12 1.6 Recurrent genomic patterns in MCL and CLL .............................................................................. 12 1.6.1 Chromosomal gain of region 3q25-q29 ......................................................................... 13 1.6.2 Chromosomal gain of region 12q13-q14 ....................................................................... 13 1.6.3 Chromosomal gain of region 18q21-q22 ....................................................................... 14 1.7 Recombinase Mediated Cassette Exchange ................................................................................ 14 1.8 Cellular model systems ................................................................................................................ 15 1.9 RNA interference ......................................................................................................................... 16 1.9.1 Mechanisms of RNAi ...................................................................................................... 16 1.9.2 Loss-of-function screen .................................................................................................. 17 1.10 Aim of the work ........................................................................................................................... 19 2 Material and Methods .......................................................................................................................... 21 2.1 Material ....................................................................................................................................... 21 2.1.1 Chemicals and biochemicals .......................................................................................... 21 2.1.2 Enzymes ......................................................................................................................... 22 2.1.3 Kits.................................................................................................................................. 23 2.1.4 Other materials .............................................................................................................. 23 2.1.5 Solutions ........................................................................................................................ 24 2.1.6 siRNA sequences ............................................................................................................ 27 2.1.7 Vectors ........................................................................................................................... 28 2.1.8 Primers ........................................................................................................................... 28 2.1.9 Antibodies ...................................................................................................................... 29 2.1.10 Cell culture ..................................................................................................................... 30 2.1.11 Tumor samples ............................................................................................................... 31 2.1.12 Instruments .................................................................................................................... 33 2.1.13 Software ......................................................................................................................... 34 2.2 Methods ...................................................................................................................................... 35 2.2.1 Cell culture ..................................................................................................................... 35 2.2.2 Transfection methods .................................................................................................... 36 2.2.3 Molecular biological standard methods ........................................................................ 37 2.2.4 Expression arrays ........................................................................................................... 42 2.2.5 Southern blot analysis .................................................................................................... 43 i Contents 2.2.6 Western blot analysis ..................................................................................................... 45 2.2.7 Fluorescence in situ hybridization .................................................................................. 46 2.2.8 Functional assays ........................................................................................................... 48 3 Results .................................................................................................................................................. 51 3.1 Transfection of suspension cells .................................................................................................